Very early life pain exposure and stress induces alterations in the developing brain and leads to altered pain sensitivity. In premature infants with a history of numerous early postnatal adverse events, behavioral responsiveness and hypothalamic-pituitary-adrenal (HPA) axis reactivity may show alterations as well.We compared a multidimensional response to a painful situation (vaccination) in three month old infants. The study involved very preterm, moderate to late preterm infants and full-term infants with varying exposure to pain and stress within the first weeks of life.At the age of three months, we evaluated the infants' reactivity to intramuscular injections for immunization.The study included 61 very preterm infants, 30 moderate to late preterm infants and 30 full-term infants.We assessed heart rate recovery, Bernese pain Score and increase of salivary cortisol following vaccination. We also evaluated the flexor withdrawal reflex threshold as well as Prechtl's General Movements. Secondly, we assessed factors potentially influencing pain reactivity such as exposure to pain/stress, gender, use of steroids or opioids and mechanical ventilation.Very preterm, moderate to late preterm and full-term infants showed different reactivity to pain in all analyzed aspects. Very preterm infants showed a lower level of behavioral and physiologic reactivity and exposure to pain/stress predicted lower cortisol increase.At three months of age, very preterm infants show an altered level of HPA axis reactivity. Efforts aiming at minimizing pain and stress in premature infants should be taken.